1. Field of the Invention
The present invention relates to a recombinant polyclonal anti-orthopoxvirus antibody (anti-orthopoxvirus rpAb), in particular a recombinant polyclonal anti-vaccinia virus antibody (anti-VV rpAb). The invention also relates to polyclonal expression cell lines producing anti-orthopoxvirus rpAb or anti-VV rpAb. Further, the application describes diagnostic and pharmacological compositions comprising anti-orthopoxvirus rpAb or anti-VV rpAb and their use in prevention, and treatment of adverse effects of vaccination, or diagnosis and treatment of orthopoxvirus infections.
2. Background Art
Smallpox is caused by airway infection with the orthopoxvirus, variola. The threat of smallpox outbreaks as a result of bioterrorism and the emergence of related viruses such as monkeypox, have revived the need for anti-orthopoxvirus therapeutics and vaccination. Vaccinia virus vaccination mediates moderate to severe adverse reactions in approximately one in every 1000. These are currently treated with anti-vaccinia virus immunoglobulin (VIG) isolated from donors with a high antibody titer. However, the estimated incidence of adverse effects resulting from a general vaccination program using live attenuated vaccinia virus exceeds the current production capacity of VIG, thereby preventing vaccination as an approach for public protection against smallpox. Furthermore, VIG has a very low specific activity resulting in a need for injection of large volumes. There is also the risk of transmission of viral diseases from serum derived VIG products, as well as problems with batch-to-batch variations. Therefore, investigations of possible alternatives for providing protecting against vaccinia virus adverse effects or infections by other orthopoxviruses have been conducted.
Orthopoxviruses produces two types infectious particles, namely the Intracellular Mature Virions (EMV) and the Extracellular Enveloped Virions (EEV). IMV plays a predominant role in host-to-host transmission and EEV plays a major role in virus propagation within the host. The IMV particle is assembled in the cytoplasm of infected cells and consists of a virally induced membrane surrounding the genome containing a homogenous core particle. EEV particles are generated by wrapping of IMV particles in a host cell-derived membrane followed by egress of the EEV particle. At a later stage the vaccinia virus infection results in cell death and release of the infectious IMV particles. Viral proteins presented at the surface of IMV or EEV particles are potential targets for antibodies, a total of five IMV-specific proteins and two EEV-specific proteins have been reported to elicit virus neutralizing and/or protective effects when used for immunization or vaccination. Additionally, neutralizing and protective effects have been observed for the passive administration of antibodies which specifically bind these proteins (summarized in Table 1).
TABLE 1AntigenVirionAntibody effectImmunization/vaccination effectA27LIMV+ neutralize11,12,15DNA: + neutralize ÷ protective6(P14)(+) protective11Protein: + neutralize + protective1,8− protective15A17LIMV+ neutralize13(P21)L1RIMV+ neutralize10,14,15DNA: + neutralize (+) protective5(P25-29)+ protective10,15Protein: + neutralize + protective2D8LIMV+ neutralize15Protein: ÷ neutralize + protective1(P32)÷ protective15H3LIMV+ neutralize4, 9(P35)A33REEV÷ neutralize10,15DNA: ÷ neutralize + protective3,5(Gp23-28)+ protective10,15,16Protein: + neutralize + protective2B5REEV+ neutralize7DNA: ÷ neutralize (+) protective3,6(Gp42)+ protective10,16Protein: ÷ neutralize + protective2,3The column “antibody effect” summarizes results from references describing the effect of an antibody reactive with the named antigen either in in vitro neutralization assays or by in vivo challenging assays to measure protectiveness. The column “immunization/vaccination effect” summarizes results from references where the antigen has been injected into animals either in protein form or as DNA. The neutralizing effect is analyzed by assessing the neutralization titer of the injected animals and the protective effect by challenging the immunized/vaccinated animals with vaccinia virus.    1. Demkowicz et al. 1992, J. Virol. 66:386-98.    2. Fogg et al. 2004, J. Virol. 78:10230-7. This reference also describes increased protection when immunization was performed with the following protein combinations B5R+A33R+L1R>A33R+L1R>A33R+B5R>B5R+L1R    3. Galmiche et al, 1999, Virology 254:71-80.    4. Gordon et al. 1991, Virology 181:671-86    5. Hooper et al. 2000, Virology 266:329-39. This reference also describes increased protection when vaccination was performed with both L1R and A33R encoding DNA.    6. Hooper et al. 2003, Virology 306:181-95. This reference also describes increased protection when vaccination was performed with the following DNA combinations: B5R+A33R+L1R+A27L and B5R+A27L, where the first combination showed better protection than the second combination.    7. Law et al. 2001, Virology 280:132-42.    8. Lai et al. 1991, J. Virol. 65:5631-5.    9. Lin et al. 2000, J. Virol. 74:3353-3365.    10. Lustig et al 2005, J. Virol. 79:13454-13462. This reference also shows enhanced protection when monoclonal antibodies against L1R, A33R and B5R were combined.    11. Ramirez et al. 2002, J. Gen. Virol. 83:1059-1067.    12. Rodriguez et al. 1985, J. Virol. 56:482-488.    13. Wallengren et al. 2001, Virology 290:143-52.    14. Wolffe et al. 1995, Virology 211:53-63.    15. U.S. Pat. No. 6,451,309 illustrates increased protection when monoclonal antibodies against L1R and A33R were combined. Further, L1R and A33R mAbs combined with at least one mAb directed against H3L, D8L, B5R, A27L and A17L is suggested, but there is no evidence of the effect of such a combination.    16. WO 03/068151 suggests individual or combinations of fully human antibodies which binds an EEV protein, in particular B5R, A33R or B7R, where B7R is a variola ortholog of B5R and shares 92.7% identity with it. The application does not contain any evidence of the neutralizing or protective effect of such compositions.
Some of the studies cited in table 1 have revealed that protection against virus challenge is generally increased when protein/DNA combinations targeting both IMV and EEV virion proteins are used for immunization/vaccination (ref. 2, 5, 6 and 10). Similarly, U.S. Pat. No. 6,451,309 illustrated that the combination anti-L1R and anti-A33R mAbs administered to mice prior to a vaccinia virus challenge had an increased protective effect compared to the individual mAbs. This correlates with early observations that vaccination with inactivated IMV particles elicited antibody responses, but did not confer protection to virus challenge in animal experiments (Boulter and Appleyard, 1973, Prog. Med. Virol. 16, 86-108).
The fact that a combination of antibodies is better than a single monoclonal antibody is further supported by the observations by Gordon et al. 1991, Virology 181:671-86, where it was shown that when comparing the neutralizing capability of a single mAb with an anti-VV envelope serum which has been purified with respect to the same antigen specificity as the mAb, or a non-purified polyclonal anti-VV envelope serum, both the purified and non-purified polyclonal anti-envelope serum was much more effective than the monoclonal antibody. Thus, both the binding of antibodies to more than one epitope on the same antigen as well as the binding of several antigens on different proteins is likely to be relevant when neutralizing vaccinia virus.